WO2002031502A1 - Chemically modified substrate and process for producing the same - Google Patents

Abstract

A chemically modified substrate with which DNAs can be efficiently explicated or stored and which is inexpensive and useful in the fields of molecular biology, biochemistry, etc.; and a method of chemical modification. The method is characterized by aminating a surface of a substrate and then carboxylating the surface with a treating fluid containing an anhydride of a polycarboxylic acid to thereby chemically modify the substrate.

Description

TECHNICAL FIELD The present invention relates to a chemically modified substrate and a method for producing the same.

 TECHNICAL FIELD The present invention relates to a chemically modified substrate capable of immobilizing a nucleic acid or a protein, which is useful in the fields of molecular biology and biochemistry.

 Light

Background technology

 Gene analysis is useful in the fields of molecular biology and biochemistry, and has recently been used in the medical field, such as disease detection.

 In gene analysis, DNA chips have recently been developed and the analysis speed has been significantly increased. However, the conventional DNA chip is a method in which a polymer such as polylysine is applied to the surface of a slide glass or a silicon substrate, and then the DNA is fixed. In addition, a method of synthesizing oligonucleotides on a glass substrate using a semiconductor technique such as photolithography has been used.

 However, in the method of immobilizing DNA by coating a polymer such as polylysine on the surface of a slide glass or silicon substrate, the immobilized state of DNA is unstable, and the DNA is peeled off in the hybrid formation process and the cleaning process. Then a problem arises. Further, there is a problem that a DNA chip using semiconductor technology is very expensive due to the complexity of the manufacturing process. In order to solve such problems, it is necessary to immobilize DNA on the surface of the substrate at high density and firmly.

Conventionally, a substrate whose surface is chemically modified is known. However, carboxylic acid activation is required to bind DNA to carboxylic acid or the like, so chemical modification of the substrate alone is not sufficient. Furthermore, as a method of introducing a carboxyl group, It is known to treat with a solution containing acid chloride. However, acid chloride is difficult to manufacture, and thus has problems in terms of stable supply, price and purity.

 An object of the present invention is to provide an inexpensive and chemically modified substrate capable of efficiently elucidating and preserving DNA in the field of molecular biology, biochemistry and the like, and a method for producing the same. It is assumed that. Another object of the present invention is to provide a substrate for stably immobilizing DNA or protein. Disclosure of the invention

 The present inventors have conducted intensive studies to achieve the above object, and as a method of chemically modifying the surface of the substrate to introduce a carboxyl group, the surface of the substrate is treated with a treatment solution containing an anhydride of polyvalent ruponic acid. Found that it would be an inexpensive way. In terms of characteristics, it was found that a lipoxyl group could be introduced to the same degree as the conventional method using an acid chloride.Claim 1 ・ The surface of the substrate was chemically modified with a polycarboxylic acid anhydride. Substrate. The substrate is silicon, glass or metal, and diamond, diamond-like carbon, graphite, hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, carbon nitride, titanium carbide, uranium carbide are coated on the surface of the substrate. And a surface treatment layer made of tungsten carbide, zirconium carbide or molybdenum carbide. The metal may be gold, silver, copper, aluminum, or tungsten.

The thickness of the surface treatment layer is 1 nm to 100 nm. Further, it has an amino group on the surface of the substrate.

 Claim 8 is a chemical modification method in which a hydrocarbon group having a carboxyl group at the terminal is bonded to the surface of the base via an amide bond, and the carboxyl group is treated with a liquid containing a polycarboxylic anhydride as a main component. This is a method for producing a substrate.

The method of treating with a solution mainly containing anhydrous polyvalent ruponic acid is preferred by immersion. A method for producing a substrate, characterized by being a method.

 When a hydrocarbon group having a terminal carboxyl group is bonded to the substrate surface via an amide bond, the primary amino group obtained by chlorinating the substrate surface and then amination and the anhydride of a polycarboxylic acid are obtained. A substrate production method characterized by reacting with a substance to form an amide bond. BEST MODE FOR CARRYING OUT THE INVENTION

 The substrate of the present invention is characterized in that the surface of the substrate has been subjected to a specific chemical modification. The chemical modification in the present invention means immobilizing a hydrocarbon having at least two or more carboxyl groups bonded to its terminal to the surface of a substrate via an amide bond. By further subjecting the chemically modified substrate to active esterification, it becomes easier to immobilize DNA or the like or a protein on the surface of the substrate.

The formic acid and acetic acid hydrocarbon groups preferably have 0 to 12 carbon atoms, and more preferably 0 to 6 carbon atoms. For example, dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, and polycarboxylic acids such as trimellitic acid can be used, and one or more acid anhydrides can be used. it can. Among them, succinic acid is preferred.

 As the active ester group bonded to the terminal of the hydrocarbon group, N-hydroxysuccinimide ester or p-ditrophenol ester is preferable.

 Such an activated ester is to immobilize a group in which an active ester group is bonded to the terminal of a hydrocarbon group via an amide bond to a substrate.

For example, when the active ester group is an N-hydroxysuccinimide ester group, the surface is chlorinated by irradiating the substrate with ultraviolet light in chlorine gas, and then aminated by irradiation with ultraviolet light in ammonia gas. Carboxylation using an acid anhydride can be carried out by dehydration-condensation of the terminal carboxyl group with carposimide or dicyclohexylcarposimide and N-hydroxysuccinimide. · The dehydration condensation in the case of employing this method will be described by way of an example. The solid support having a surface chemically modified and having a carboxyl group can be converted into a carbohydrate or hexylcarbodiimide, and N-- It is immersed in 1,4-dioxane solution in which hydroxysuccinimide or p-nitrophenol is dissolved, washed and dried. In this way, a substrate to which a hydrocarbon group having an N-succinimide ester group or a p-nitrophenol ester group at the terminal is obtained.

 This method was first developed by the present inventors. By making the surface of the substrate chemically modified with an activated ester group, the user can easily use it to immobilize DNA directly on the surface of the substrate by amide bonds. In the present invention, the substrate to be chemically modified as described above includes metals such as diamond, gold, silver, copper, aluminum, tungsten, and molybdenum, a laminate of the above metals and ceramics, and plastics such as polycarbonate and fluororesin. And the like.

 Other materials can be used as long as they are chemically stable, such as graphite and diamond-like carbon. Further, a mixture of a plastic and the above-mentioned metal, ceramics, diamond or the like may be used. Furthermore, on a glass slide or silicon, diamond, diamond carbon, graphite, hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, ichhitorium carbide, titanium carbide, uranium carbide, tungsten carbide, zirconium carbide or molybdenum carbide, etc. What formed the surface treatment layer may be used. The thickness of the surface treatment layer is preferably from 1 nm to 100 nm.

 Among them, diamond or diamond-like carbon is preferred from the viewpoints of remarkably high solidification density of DNA and the like and thermal conductivity.

As the material of the diamond substrate, any of synthetic diamond, high pressure formed diamond, natural diamond and the like can be used. Also, their structures may be single crystals or polycrystals, or may be misaligned. Microwave plasma CVD, It is preferable to use diamond or diamond-like carbon produced by high-frequency plasma CVD, ion vapor deposition, arc vapor deposition, or the like. The method for forming the substrate of the present invention can be performed by a known method. For example, microwave plasma CVD, ECRCVD, IPC, DC sputtering, ECR sputtering, ion plating, arc ion plating ', EB evaporation, resistance heating evaporation, and the like. In addition, there may be mentioned those obtained by mixing a metal powder or a ceramic powder with a resin as a binder to form a bond. Further, a material obtained by pressing a raw material such as a metal powder or a ceramic powder using a press molding machine and sintering the material at a high temperature can also be used.

 It is desirable that the substrate surface of the substrate of the present invention is intentionally roughened. This is because such a roughened surface is advantageous in increasing the surface area of the substrate and immobilizing a large amount of DNA and the like. The shape of the substrate is not particularly limited, such as a flat plate, a thread, a sphere, a polygon, and a powder.

 Further, the diamond substrate may be a composite of diamond and another substance (for example, a two-layer substrate). Example

 Hereinafter, the present invention will be described with reference to examples.

Example 1

 The surface was chlorinated by irradiating a 3 mm square CVD diamond with ultraviolet light in chlorine gas, and then aminated by irradiation with ultraviolet light in ammonia gas, followed by carboxylation with anhydrous dicarboxylic acid. This carboxylation is performed by dissolving 0.2 g of succinic anhydride in 42 mL of pyrrolidone and further immersing the aminated substrate for 20 minutes in a treatment solution containing 1 mL of 1 mol of sodium borate (pH 8.0). Gently remove the substrate. Then, the carboxylation is completed by washing with water.

The surface-modified substrate was treated with 2.5 mg / m 1 of carposimide and N-hydride. Roxysuccinimide 1.5 mg / m 1 dissolved in 1,4-dioxane solution (an amount of 10 O / i 1 per 3 mm square diamond) for 15 minutes to obtain a terminal carboxyl group Was subjected to dehydration condensation. After completion of the reaction, the substrate was washed with water, further washed with a 1,4-dioxane solution, and dried to obtain a chemically modified substrate.

After immobilizing the 20-mer oligonucleotide using the substrate prepared in Example 1 described above, a hybrid was formed with a fluorescent-labeled probe having a complementary sequence, and the oligonucleotide was immobilized using a fluorometer. Estimated quantity. As a result, in Example 1, it was 9 pmo 1 per 3 mm square substrate, and in Example 2, it was 0.1 pm o 1 per 1 mm ² , indicating that the oligonucleotides were fixed at high density in each case. It was revealed. Industrial applicability

 Since the chemically modified substrate of the present invention is chemically modified and has an activated ester group, it can stably immobilize nucleic acids such as DNA, and is used for PCR or as a DNA array substrate. This is advantageous. In addition, since the introduction of the carboxy group is performed using a solution containing a polycarboxylic anhydride as a main component instead of acid chloride, the substrate is inexpensive and can be stably immobilized. Manufacturing method.

Claims

The scope of the claims  1. A substrate whose surface is chemically modified with a polycarboxylic acid anhydride.  2. The chemically modified substrate according to claim 1, wherein the substrate is silicon, glass or metal.  3. The substrate according to claim 2, wherein the metal is gold, silver, copper, aluminum or tungsten.  4. The substrate according to any one of claims 1 to 3, wherein the substrate is provided with a surface treatment layer made of diamond, diamond-like carbon or graphite on the surface.  5.'The substrate has a surface treatment layer formed of hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, trim carbide, titanium carbide, uranium carbide, tungsten carbide, zirconium carbide or molybdenum carbide formed on the surface. A substrate which has been subjected to the chemical modification according to claim 1.  6. The chemically modified substrate according to claim 4, wherein the thickness of the surface treatment layer is 1 nm to 100 nm.  7. The chemically modified substrate according to claim 1, wherein the substrate has an amino group on the surface.  8. Chemical modification by a method in which a hydrocarbon group having a carboxyl group at the terminal is bonded to the surface of the base via an amide bond, and the carboxyl group is treated with a liquid containing a polycarboxylic anhydride as a main component. A method for producing a substrate.  9. The method for producing a chemically modified substrate according to claim 8, wherein the method of treating with a liquid containing an anhydride of a polycarboxylic acid as a main component is a method by immersion. 10. When a hydrocarbon group having a terminal carboxyl group is bonded to the substrate surface via an amide bond, the surface of the substrate is chlorinated and then aminated to obtain an anhydride of a primary amino group and a polycarboxylic acid. To form an amide bond by reacting 10. A method for producing a chemically modified substrate according to claim 8 or 9.